Localization of endocytic proteins in cells expressing Pan1-FKBP and Mito-FRB-mCherry, and Actin-dependent recruitment of Prk1-GFP to the Pan1p-anchoring peroxisome. (A) Relative localization of Pan1p at cortical patches in cells anchoring Pan1p to the perosisome. Data show the mean ± SD from three experiments (n = 30 patches for each experiment). ***, P value < 0.001, unpaired t test with Welch’s correction. (B) Localization of GFP-tagged endocytic proteins and Mito-FRB-mCherry before rapamycin addition. (C) Localization of GFP-tagged Yap1801p or Ede1p and Mito-FRB-mCherry before and at 30 min after rapamycin addition. (D) Fluid-phase endocytosis in cells expressing Mito-GFP-FRB and Pan1-FKBP. Cells were labeled with 200 μM FM4-64 for 15 min on ice. The images were acquired at 5 and 10 min after FM4-64 internalization. Representative fluorescence intensity profiles along a line are indicated in the right panels. (E) Schematic diagram of the inducible peroxisome redistribution assay. Pan1-FKBP was co-expressed with Pex-FRB-mCherry in cells expressing GFP-tagged Prk1p, Srv2p, or Cof1p. Addition of rapamycin causes heterodimerization of FRB and FKBP and thereby recruits Pan1p and its binding protein to the peroxisome surface. (F) Localization of Prk1-GFP and Pex-FRB-mCherry before or 30 min after rapamycin addition. Cells were grown to log phase at 25°C. White arrowheads indicate examples of co-localization. Right image pair was acquired 30 min after rapamycin addition in the presence of 250 μM LatA. Scale bar, 2.5 μm.